Antibiotic TA is a hybrid polyketide/non-ribosomal peptide with an unusual alkylation pattern. Because alkylation has been shown in several cases to increase the activities of bioactive compounds, understanding the alkylation strategy utilized in the biosynthesis of antibiotic TA would be relevant to efforts to engineer novel alkylated therapeutic candidates. I hypothesize that the C13 ethyl and a methyl precursor to the C17 methoxymethyl in antibiotic TA are installed by a recently characterized isoprenoid-like alkylation strategy. I further hypothesize that the C17 methyl precursor undergoes a hydroxylation/O-methylation sequence to generate the final methoxymethyl structure. This hypothesis is based on several observations. First, the locations of the alkyl branches are consistent with an isoprenoid-like origin from 2-ketothioester biosynthetic intermediates. Second, the antibiotic TA biosynthetic cluster contains homologs of a cassette of proteins responsible for the methylation of a Bacillus subtilis secondary metabolite by a similar strategy. Finally, feeding studies utilizing isotopically labeled precursors are consistent with the proposed alkylation pathway. The specific aims of the proposed research are to reconstitute the alkylation sequence on model substrates in vitro using the antibiotic TA biosynthetic proteins expressed heterologously in E. coli, and to identify the components of the pathway that discriminate between the two alkylation sites and determine branch identities. Relevance The proposed research is relevant to public health in that it will aid access to therapeutic candidates that would otherwise be too complex to synthesize chemically. By characterizing the proteins involved in the production of antibiotic TA, it may be possible to use these proteins to engineer new, more effective analogs of already existing drugs. [unreadable] [unreadable] [unreadable]